Technical Field
The present invention generally relates to methods and kits for selectively amplifying, detecting or quantifying a DNA fragment with a specific end sequence, especially that created by a restriction enzyme, in a sample containing other DNA fragments.
Description of the Related Art
The discovery of cell-free fetal nucleic acids in the plasma of pregnant women has opened up new possibilities for noninvasive prenatal diagnosis and monitoring (Lo et al., Lancet, 350:485-7 (1997); Lo and Chiu, Nat Rev Genet, 8:71-77 (2007)). Over the past decade, a list of described fetal-specific markers have expanded from gender-dependent Y-chromosomal sequences (Lo et al., Am J Hum Genet, 62:768-75 (1998); Lo et al., Clin Chem, 45:1747-51 (1999); Leung et al., Lancet, 352:1904-5 (1998); and Rijnders et al., Prenat Diagn, 23:1042-4 (2003)) and paternally-inherited polymorphic markers (Lo et al., N Engl J Med, 339:1734-8 (1998); Saito et al., Lancet, 356:1170 (2000); Chiu et al., Lancet, 360:998-1000 (2002); and Chow et al., Clin Chem, 53:141-2 (2007)) to universal fetal markers such as placental-specific mRNA transcripts (Ng et al., Proc Natl Acad Sci USA, 100:4748-53 (2003); Tsui et al., J Med Genet, 41:461-7 (2004); and Lo et al., Nat Med, 13:218-223 (2007)) and those dependent on placental-specific epigenetic signatures (Chim et al., Proc Natl Acad Sci USA, 102:14753-8 (2005); Tong et al., Clin Chem, 52:2194-202 (2006); and Chan et al., Clin Chem, 52:2211-8 (2006)). In addition to clinical applications, such as prenatal gender determination and prenatal RHD genotyping, researchers have recently demonstrated that trisomy 21 and trisomy 18 pregnancies could be inferred by the analysis of fetal nucleic acids in maternal plasma samples (Lo and Chiu, Nat Rev Genet, 8:71-77 (2007)).
The tumor suppressor gene SERPINB5 (maspin), which is expressed in placenta (Dokras et al., Placenta, 23:274-80 (2002)) and exhibits a tissue-specific methylation pattern (Futscher et al., Nat Genet, 31:175-9 (2002)), is the first universal fetal epigenetic marker reported (Chim et al., Proc Natl Acad Sci USA, 102:14753-8 (2005)). To detect hypomethylated placental SERPINB5 in an excess background of hypermethylated SERPINB5 of maternal origin in a maternal plasma sample, the use of bisulfite treatment was reported (Frommer et al., Proc Natl Acad Sci USA, 89:1827-31 (1992); and Herman et al., Proc Natl Acad Sci USA, 93:9821-6 (1996)). However, it was documented that up to 96% of the DNA in the plasma sample would be degraded by the bisulfite treatment (Grunau et al., Nucleic Acids Res, 29:E65-5 (2001)), leaving a minute amount of DNA for analysis. In a recent study, Chan et al. have described the discovery and possible utility of another universal fetal marker in maternal plasma (Chan et al., Clin Chem, 52:2211-8 (2006)). This marker, the RASSF1A tumor suppressor gene, exhibits an opposite methylation pattern in the placenta and maternal blood cells to the SERPINB5 gene (Chiu et al., Am J Pathol, 170:941-50 (2007)). In this case, methylation analysis involving the use of methylation-sensitive restriction endonucleases could be employed, thus cutting the hypomethylated RASSF1A from maternal blood cells and leaving the hypermethylated placental RASSF1A intact for amplification.
If the same approach were to be applied to the detection of the placenta-derived hypomethylated SERPINB5 molecules, an endonuclease which exhibits an opposite action to the methylation-sensitive restriction endonucleases described above would be needed. In the current literature, it has been reported that the McrBC enzyme, which is an endonuclease, would cleave methylcytosine-containing DNA sequences (Sutherland et al., J Mol Biol, 225:327-48 (1992)). However, not much is known about the exact molecular action of this enzyme, and the cleavage spacing can range from 32 basepairs to two kilobases long (Stewart et al., Biol Chem, 379:611-6 (1998)). For experiments which require high precision, as in the case of detecting fetal DNA in maternal plasma, such an enzyme would not be reliable.
A potential approach would be to develop a specific method for the detection of the restriction products of hypomethylated SERPINB5 sequences following a methylation-sensitive restriction endonuclease treatment. To allow this to be done, a new method which can specifically detect a shorter DNA fragment (i.e. restricted) in the presence of an excess of longer DNA fragments (i.e. unrestricted) is needed.